cancers Article Epithelial-to-Mesenchymal Transition Enhances Cancer Cell Sensitivity to Cytotoxic Effects of Cold Atmospheric Plasmas in Breast and Bladder Cancer Systems Peiyu Wang 2,3 , Renwu Zhou 4 , Patrick Thomas 2,3,5 , Liqian Zhao 6, Rusen Zhou 4, Susmita Mandal 7, Mohit Kumar Jolly 7, Derek J. Richard 2,3, Bernd H. A. Rehm 8, Kostya (Ken) Ostrikov 9, Xiaofeng Dai 1,*, Elizabeth D. Williams 2,3,4 and Erik W. Thompson 2,3 1 Wuxi School of Medicine, Jiangnan University, Wuxi 214122, China 2 Queensland University of Technology (QUT), School of Biomedical Sciences, Brisbane 4059, Australia 3 Translational Research Institute, Woolloongabba 4102, Australia 4 School of Chemical and Biomolecular Engineering, The University of Sydney, Sydney 2006, Australia 5 Queensland Bladder Cancer Initiative (QBCI), Woolloongabba 4102, Australia 6 The First School of Clinical Medicine, Southern Medical University, Guangzhou 510515, China 7 Centre for BioSystems Science and Engineering, Indian Institute of Science, Bangalore 560012, India 8 Centre for Cell Factories and Biopolymers, Griffith Institute for Drug Discovery, Griffith University, Nathan 4111, Australia 9 School of Chemistry and Physics, Queensland University of Technology, Brisbane 4000, Australia; Citation: Wang, P.; Zhou, R.; * Correspondence: [email protected] Thomas, P.; Zhao, L.; Zhou, R.; Mandal, S.; Jolly, M.K.; Richard, D.J.; Simple Summary: Cold atmospheric plasma (CAP) and plasma-activated medium (PAM) are known Rehm, B.H.A.; Ostrikov, K.; et al. to selectively kill cancer cells, however the efficacy of CAP in cancer cells following epithelial- Epithelial-to-Mesenchymal Transition mesenchymal transition (EMT), a process which endows cancer cells with increased stemness, Enhances Cancer Cell Sensitivity to metastatic potential, and resistance to conventional therapies, has not been previously examined. We Cytotoxic Effects of Cold have used several established models of EMT to show that PAM is significantly more active in cancer Atmospheric Plasmas in Breast and cells exhibiting EMT than their epithelial counterparts. We further show that this enhancement Bladder Cancer Systems. Cancers correlated with increased levels of reactive oxygen species (ROS) in the mesenchymally-shifted 2021, 13, 2889. https://doi.org/ cell lines. 10.3390/cancers13122889 Academic Editors: Annemie Bogaerts, Abstract: Cold atmospheric plasma (CAP) has emerged as a highly selective anticancer agent, most Katharina Stapelmann and recently in the form of plasma-activated medium (PAM). Since epithelial–mesenchymal transition Abraham Lin (EMT) has been implicated in resistance to various cancer therapies, we assessed whether EMT status is associated with PAM response. Mesenchymal breast cancer cell lines, as well as the mes- Received: 8 April 2021 enchymal variant in an isogenic EMT/MET human breast cancer cell system (PMC42-ET/LA), were Accepted: 29 May 2021 more sensitive to PAM treatment than their epithelial counterparts, contrary to their responses to Published: 9 June 2021 other therapies. The same trend was seen in luminal muscle-invasive bladder cancer model (TSU- Pr1/B1/B2) and the non-muscle-invasive basal 5637 bladder cancer cell line. Three-dimensional Publisher’s Note: MDPI stays neutral spheroid cultures of the bladder cancer cell lines were less sensitive to the PAM treatment compared with regard to jurisdictional claims in to their two-dimensional counterparts; however, incrementally better responses were again seen published maps and institutional affil- in more mesenchymally-shifted cell lines. This study provides evidence that PAM preferentially iations. inhibits mesenchymally-shifted carcinoma cells, which have been associated with resistance to other therapies. Thus, PAM may represent a novel treatment that can selectively inhibit triple-negative breast cancers and a subset of aggressive bladder cancers, which tend to be more mesenchymal. Our approach may potentially be utilized for other aggressive cancers exhibiting EMT and opens new Copyright: © 2021 by the authors. opportunities for CAP and PAM as a promising new onco-therapy. Licensee MDPI, Basel, Switzerland. This article is an open access article Keywords: cold atmospheric plasma (CAP); plasma-activated medium (PAM); epithelial–mesenchymal distributed under the terms and transition (EMT); reactive oxygen species (ROS) conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Cancers 2021, 13, 2889. https://doi.org/10.3390/cancers13122889 https://www.mdpi.com/journal/cancers Cancers 2021, 13, 2889 2 of 22 1. Introduction Cold atmospheric plasma (CAP) represents an emerging onco-therapeutic approach offering a new opportunity to effectively manage aggressive cancers [1,2]. CAP is generated under atmospheric pressure conditions at room temperature, and is a source of charged particles, free radicals, metastable species, UV radiation and electric fields [3]. In addition to the direct CAP strategy, CAP activity can also be transferred to liquids (i.e., plasma activated medium (PAM)), adding flexibility to the application of plasma therapy [4–6]. PAM, produced by activating culture medium with CAP, has been found to selectively cause a significant reduction in the viability of cancer cells, including breast cancer [7], bladder cancer [8], osteosarcoma [9], and skin cancer [10] cells. Epithelial-mesenchymal transition (EMT) is an important cellular process during organ development through which cells lose their epithelial features at the cellular and molecular levels, and acquire mesenchymal traits such as stellate morphology, reduced adhesion, and increased migration [11]. EMT processes are hijacked by malignant epithelial cells, leading to increased mobility and invasion, and in some cases, reduced proliferation [12]. Successful metastasis also requires MET to revert the phenotype to an epithelial state [13]. We used several EMT model systems available in breast and bladder cancer to explore the relationship between EMT status and CAP response in this study. We and others have previously shown that intrinsic molecular subgroups of human breast cancer cell lines differed in terms of EMT state, with the most aggressive Basal B/Mesenchymal subgroup being more mesenchymal than the Basal/Basal A, which in turn was more mesenchymal that the Luminal subgroup [14–17]. Some of these cell lines have also been aligned with newly defined clinical subgroups of triple negative breast cancers (TNBCs) [18–20]. We have examined the PAM responses of MCF-7 (Luminal and ER/PR+), MDA-MB-468 (Basal A/Basal-Like 1 (BL1), more epithelial and TNBC) and MDA-MB- 231 (Basal B /Mesenchymal (M)–more mesenchymal and TNBC), as well as the isogenic breast cancer PMC42-ET/LA model, in which the epithelial PMC42-LA cell line arose spontaneously from the parental, mesenchymal PMC42-ET [21–24], although both map tentatively to the Basal B intrinsic classifier [24]. We also assessed bladder cancer cell lines that represent non-muscle invasive bladder cancer (NMIBC; 5637 cells derived from basal bladder cancer and relatively epithelial [25]) and muscle invasive bladder cancer (MIBC; T24/TSU-Pr1 cells derived from high grade transitional cell carcinoma [2]), as well as TSU-Pr1-derived sublines (TSU-Pr1-B1, -B2). The TSU-Pr1-derived B1 and B2 sublines were selected for increased metastatic colonization of mouse bones and exhibit sequential increases in epithelial phenotype [26,27]. Collectively these models provide an opportunity to assess the relationship between EMT and PAM response in two different tumor streams. 2. Materials and Methods 2.1. Reagents CellROX® Orange (C10443, Thermo Fisher Scientific, San Diego, CA, USA), Hoechst 33342 (H3570, Themo Fisher Scientific), Happy Cell Advanced Suspension Matrix (HC- ASM; Vale Life Sciences, Biocroi, Ireland), CellEvent™ Caspase-3/7 Green ReadyProbes™ Reagent (C10423, Thermo Fisher Scientific), Cell CellTiter-Glo® 3D Cell Viability Assay (Promega, Madison, WI, USA) 2.2. PAM Generation The CAP device used in this study was a typical commercial argon plasma jet (model kINPen 09, INP Greifswald, Germany) [28]. During the experiment, pure argon gas was kept at the flow rate of 5.0 Standard Liter per Minute. Aliquots of 1.5 mL medium containing serum in 2 mL centrifuge tubes were activated by CAP for 10 min and named ‘10PAM’ here. The distance between the plasma jet nozzle to the medium surface was 10 mm. PAM was then diluted to different concentrations, designated by the percent remaining (e.g., 70% 10PAM refers to 70% concentration at use). After treatment, different concentrations of PAM were used in cultured cells, which were seeded in 96-well plates. Cancers 2021, 13, 2889 3 of 22 2.3. Cell Culture Human breast cancer cell lines MCF-7, MDA-MB-468, and MDA-MB-231, originally from the American Type Culture Collection (ATCC; Manassas, VA, USA) were transferred from the Lombardi Cancer Center, USA. Parental PMC42-ET cells and the spontaneously de- rived, epithelially shifted PMC42-LA cells were previously described [24]. Human bladder cancer cell line 5637 (ATCC®HTB-9™) was purchased from the ATCC. The T24/TSU-Pr1 cells are as previously described [26,27]. Cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Thermo Fisher Scientific, Melbourne, VIC, AU) supple- mented with 10% foetal bovine serum (FBS, Gibco Thermo Fisher Scientific, AU) (MCF-7, MDA-MB-468, MDA-MB-231 and TSU-Pr1/B1/B2), or Roswell Park Memorial Institute (RPMI)-1640 (Gibco Thermo